LGR6 is a potential diagnostic and prognostic marker for esophageal squamous cell carcinoma

Abstract Background Leucine‐rich repeat–coupled receptor 6 (LGR6) is a marker of the skin, nails, and other types of adult tissue stem cells and has been widely found to be related to the development and progression of a variety of cancer types. The clinical significance and biological function of LGR6 in esophageal squamous cell carcinoma (ESCC) have not been determined. Methods The expression of LGR6 at the transcriptional level was analyzed by searching the TCGA and UCSC data sets. Immunohistochemistry, WB, and q‐PCR were used to detect the expression of LGR6 in ESCC and adjacent normal tissues. LGR6 PPI networks and KEGG pathways were used to analyze the potential biological functions of LGR6. Results The expression of LGR6 in ESCC tissues was significantly higher than that in normal tissues and was negatively correlated with the differentiation degree of ESCC and the prognosis of the patients but not closely correlated with the TNM stage of ESCC. PPI networks showed that LGR6 had a close interaction with RSPO1, RSPO2, RSPO3, and RSPO4. KEGG pathway analysis showed that LGR6 activated the Wnt/β‐catenin signaling pathway by binding with RSPO ligands to promote the progression of ESCC. Conclusion LGR6 can serve as a potential diagnostic and prognostic marker for ESCC.

molecular biomarkers associated with the clinical factors of esophageal cancer is critical. 3 Leucine-rich repeat-containing G protein-coupled receptors (LGRs) are a subgroup of the seven-transmembrane G protein-coupled superfamily that regulates various physiological processes associated with various diseases, 4 and its member LGR4-6 has high homology. 5 Many studies have recently explored the biological functions of LGR4-6 in various human cancer types. [6][7][8][9][10][11][12] LGR4-6 plays an important role in activating the Wnt/β-catenin pathway by binding with R-spondin (RSPO) ligands, which are closely related to tumor progression and invasion. [10][11][12] LGR6 plays a pivotal role in adult stem cells, which are markers of various types of adult stem cells in the skin, nails, and a group of basal and intraluminal progenitors that induce luminal tumorigenesis. 13,14 LGR6 can promote the self-renewal and progression of nonsmall-cell lung cancer and has strong carcinogenic potential. 15 LGR6 is highly expressed in gastric and colon cancer tissues and is associated with local tumor growth. 16,17 Recent studies have shown that LGR6 enhances stemness and chemoresistance in ovarian cancer cells by activating the Wnt/β-catenin signaling pathway. 18 However, to date, there has been no research on the correlation between the expression of LGR6 at the protein level and clinical-pathological factors and prognostic features in patients with ESCC.
In this study, we evaluated the clinical significance of LGR6 expression in 102 patients with ESCC and analyzed its association with clinicopathological features and patient survival. We explored the functional effects and potential molecular mechanisms of LGR6 on ESCC progression by using the University of California Santa Cruz (UCSC) and the Cancer Genome Atlas (TCGA) data, constructing a protein-protein interaction (PPI) network and performing Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.

| RNA extraction, reverse transcription, and real-time quantitative PCR
Total RNA was extracted from frozen tissue using TRIzol reagent (Ambion) according to the manufacturer's instructions, and 1 mg of RNA was reverse transcribed for first complementary DNA strand synthesis using a miScript Reverse Transcription Kit (Qiagen). Realtime quantitative PCR was performed using a SYBR Premix EX Taq Kit (Takara). The relative mRNA expression of LGR6 was detected with the 2−ΔΔct method using specific primers, and its expression level was normalized to that of endogenous β-actin. All primers were designed by BioSune Biotechnology Co., Ltd. The sequences of the primers used are as follows: LGR6: 5′-TGGGGAACCCTCTGCTACAG-3′ (forward) and 5′-CAGGTACTGGAATGCCGATCT-3′ (reverse); and β-actin: 5′-CTCCATCCTGGCCTCGCTGT-3′ (forward) and 5′-GCTGTCACCTTCACCGTTCC-3′ (reverse).

| Western blot analysis
Tissues were lysed in Western and radioimmunoprecipitation assay

| Immunohistochemistry (IHC)
ESCC and adjacent normal tissues from 102 patients were fixed with formalin and embedded in paraffin, followed by IHC studies using a human anti-LGR6 antibody (1:200, Abcam). The degree of LGR6 staining was calculated and quantified according to the following aspects: score of stained tumor cells (0, ≤5% positive stained cells; 1, 5%-25% positive stained cells; 2, 26%-50% positive stained cells; 3, 51%-75% positive stained cells; and 4, ≤75% positive stained cells) multiplied by the staining intensity score (0, no staining; 1, weak staining, light yellow; 2, moderate staining, yellowish brown; and 3, strong dye, brown) to obtain the final score. A final score of 3 or lower was classified as low expression, and a final score of 4-12 points was classified as high expression.
The above scoring process was completed by two pathologists (Xinjian Lin and Wannan Chen) in an independent blinded manner.
Before an agreement was reached, the two pathologists discussed any inconsistent scores.

| LGR6 RNA-seq analysis based on UCSC and TCGA data
Differences in the mRNA expression of LGR6 in different normal tissues, as well as in different cancer tissues and the corresponding adjacent normal tissues, were investigated by searching the UCSC (http://genome.ucsc.edu/) and TCGA (http://cance rgeno me.nih.gov) databases.

| PPI network construction
Using the Search Tool for the Retrieval of Interacting Genes (STRING) database (http://www.string-db.org/), a PPI network related to LGR6 was established. The interactions procured included known interactions and predicted interactions.

| KEGG pathway analysis
The signaling pathways involving LGR6 were determined with the KEGG pathway database (https ://www.kegg.jp/kegg/pathw ay.html), and the function of the LGR6 gene was analyzed.

| Statistical analysis
Statistical analysis was performed using SPSS 24 TA B L E 1 Association between the expression of LGR6 and clinicopathological factors (n = 102) differences between each of the two groups. All data are expressed as the mean ± standard deviation (SD) of three independent assays.
The association between LGR6 expression and the clinicopathological features of ESCC patients was analyzed using the Pearson chi-square test. The survival curve was plotted using the Kaplan-Meier method. When P < .05, the difference was considered to be significant. We used IHC to study LGR6 expression in ESCC tissues and the corresponding adjacent normal tissues (NT) in 102 patients.

| LGR6 expression is significantly higher in ESCC tissues than in normal tissues
Representative staining images of LGR6 expression are shown in Figure 4A. Positive immunoreactivity in ESCC was mainly localized in the cell membrane, cytoplasm and extracellular matrix, and the cell membrane, cytoplasm and extracellular matrix were rarely stained in normal tissues. The expression of LGR6 was significantly higher in ESCC tissue than its corresponding adjacent normal tissue (paired t test P < .001; Figure 4B; Table 2). The lower the differentiation level of the ESCC was, the higher the expression of LGR6 was ( Figure 4C).

| LGR6 expression is associated with ESCC patient survival and prognosis
We analyzed the association between LGR6 expression and clini- LGR6 mRNA expression in the artery-aorta median is the highest, and its expression in esophageal tissue is relatively low tumor node metastasis category (T, P = .478; N, P = .576), or clinical stage (P = .112).
LGR6 expression was significantly negatively correlated with 5-year survival (P < .001) and differentiation status (Table 1). Kaplan-Meier survival analysis showed that the 5-year survival of ESCC patients with high LGR6 expression was significantly lower than that of ESCC patients with low LGR6 expression (log-rank test, P < .001; Figure 5).   Figure 6B shows the first ten proteins that interact with LGR6: RSPO1, RSPO2, RSPO3, RSPO4, ZNRF3, RNF43, LGR4, LGR5, UBC, and UBB. The minimum required interaction score was 0.905 (very high confidence). RSPO1, RSPO2, RSPO3, and RSPO4 (interaction score ≧0.967) are activators of the canonical Wnt signaling pathway by acting as a ligand for LGR4-6 receptors. However, our KEGG pathway analysis also showed that LGR6 functions primarily through its participation in the Wnt/β-catenin signaling pathway (Figure 7).

| D ISCUSS I ON
The malignancy of esophageal cancer is high, and its prognosis is poor. Predictive markers and effective molecular therapy targets are urgently needed to improve the prognosis of patients with esophageal cancer.
LGR6 has been widely reported to be an important stem cell marker in many cancer types that promotes the occurrence and progression of cancer. 19,20 Paradoxically, LGR6 has been reported to act as a tumor suppressor gene in colon and breast cancers. 21 suggest that LGR6 may be a potential therapeutic target for ESCC, providing a potential therapeutic strategy by increasing the degradation or reducing the expression of LGR6 to interfere with tumor progression.
The limitation of this study is that there was no in-depth study of the molecular mechanisms of LGR6 in the development and progression of esophageal cancer, which is where further research and exploration are needed in the future.
In summary, this study confirmed for the first time that LGR6 is highly expressed in ESCC tissues and that increased expression of LGR6 is associated with a poor prognosis of ESCC patients.
LGR6 may promote ESCC progression by activating the Wnt/β-catenin signaling pathway. These findings provide a basis for the potential application of LGR6 as a biomarker for early diagnosis and as a target gene for early therapeutic intervention.

CO N FLI C T S O F I NTE R E S T
The authors have no conflicts of interest to disclose.